Microfluidic Structures for Biomedical Applications

ABSTRACT

Microfluidic structures featuring substantially circular channels may be fabricated by embossing polymer sheets.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of, and incorporatesherein by reference in its entirety, U.S. Provisional Patent ApplicationNo. 60/973,529, which was filed on September 19, 2007.

TECHNICAL FIELD

In various embodiments, the invention relates to microfluidic structuresfor biomedical applications and, more particularly, to microfluidicconstructs with a vascular-like geometry for in vitro, in vivo, and exvivo applications in biomedical and clinical research and therapy.

BACKGROUND

Existing structures for blood flow, such as hollow fibers, tubing, andmachined structures are typically produced using conventional macroscalechemical and mechanical processing techniques. Microfluidic devices aretypically fabricated using conventional lithographic or etchingtechniques combined with replica molding. The methods for generatingthese structures that attempt to mimic vascular networks typicallysuffer from difficulties in reproducing, on the microscale, the specificand vitally important features of blood vessels. Methods using hollowfibers or other tubular structures and machined orstereolithographically formed elements for therapeutic devices, such asrenal dialysis cartridges, liver assist devices, and pulmonary supportdevices, are typically limited in terms of the minimum diameterachievable. In addition, vessel bifurcations in tube and fiber-basedconstructs typically contain sharp angles due to limitations in theassembly processes. These limitations also introduce sudden changes indiameter at vessel diameter expansions and contractions. The sharp andsudden non-physiologic features lead to disturbed flow and poor controlover key parameters, such as wall shear stress, leading to increasedlevels of inflammation and clotting and difficulties in seeding cellsalong the walls of the channels.

Microfluidic devices have addressed some of the shortfalls associatedwith fiber, tubing, and machining or solid freeform techniques.Principally, microfluidics has enabled a dramatic reduction in minimumfeature size and simplification of the assembly processes. However,existing microfluidic fabrication techniques typically do not enablesmooth transitions at bifurcations or vessel diameter changes, becausethe processes used to build the microfluidic master molds often resultin rectangular geometries and do not enable tapered transition regions.

Conventional techniques for microfluidics include the use oflithographically-formed master molds (conventional positive or negativephotoresist or SU-8 epoxy resin) that produce rectangular ornearly-rectangular sidewalls. Techniques have been reported that producecurvature in the sidewalls using JSR photoresist or other photoresistscombined with baking processes to slump the sidewalls. These processesare not well-controlled and do not produce an inverse-circular geometryneeded for replica molding. In addition, these processes do not enablesmooth transitions at bifurcations or smooth changes in vessel diameterbecause they are essentially layer-by-layer deposition and exposuretechniques, and therefore by nature they result in step changes ingeometry.

Etching processes such as plasma etching or wet etching typically havethe same limitations as do the lithographic techniques. Deep ReactiveIon Etching (DRIE) techniques are highly anisotropic, and, when modifiedto produce graded sidewalls, still produce straight walls and sharpcomers. Isotropic RIE techniques and isotropic wet etching techniquestypically have very slow etch rates and are not well-controlled forlonger etch times and arbitrary geometries. In addition, isotropicetching does not maintain a circular aspect ratio for deeper etchingprocesses.

SUMMARY OF THE INVENTION

In various embodiments, the present invention utilizes a microfluidicprocess sequence in which master molds are formed and then polymersheets are replica molded from the master molds and joined together toform vascular networks. Large-scale three-dimensional structures may beproduced by layering the microfluidic network sheets together withfluidic manifold connections between the layers. One difference betweenthe exemplary methods described herein and the former methods is theability, using the methods described herein, to form smooth,vascular-like geometries of microchannel networks in three dimensions.In some embodiments, this is enabled by the nature of the master moldformation process, as well as the related assembly methods for thefluidic manifold joining layers together. In certain embodiments of thepresent invention, master fabrication techniques result in trulysemicircular geometries, as well as smooth transitions at vesseldiameter changes and at vessel bifurcations. Manifold structuring andassembly techniques capable of providing similar smooth flow transitionsare used to complete the three-dimensional fluidic network.

Exemplary commercial applications for embodiments of the presentinvention include the use of the microvascular network structures astools for basic research in cardiovascular, cancer, and organ-specificdisease biology, drug discovery, and drug safety testing. In addition,embodiments of the present invention find commercial application inorgan assist devices for liver, kidney, lung, and other vascularizedorgans and tissues, as well as in organ replacement structures.

Exemplary advantages of the methods and devices described herein includethe ability to construct smooth transitions at vessel bifurcations andat vessel diameter changes in a manner similar to healthy physiologicstructures, which are scalable to large three-dimensional structures, aswell as the ability to produce substantially cylindrical microchannelgeometries. Other advantages are that embodiments of the presentinvention are much more physiologically realistic in structure. Formermethods are forced to choose between employing large-diametercylindrical tubes or smaller diameter microfluidic structures withapproximately rectangular geometries, while embodiments of the presentinvention enable substantially cylindrical geometries at any size scale.In addition, former structures include sharp angles, dead spaces, andsudden expansions/contractions between vessels at bifurcations and atdimensional changes found in microvascular networks. Embodiments of thepresent invention enable smooth transitions at bifurcations and atchanges in diameter, minimal dead volume, and smooth flow. Thus,disturbed flow conditions associated with thrombus formation areminimized or avoided, plaque formation and inflammatory response areminimized or avoided, and substantially uniform wall shear stress atevery point along the inner circumference of the lumen is achieved.

In one aspect, embodiments of the invention feature a method forfabricating a microfluidic structure having a channel with substantiallycircular cross section. Herein, a cross section is understood to beperpendicular to a longitudinal axis of the channel, and the point wherethe longitudinal axis intersects with the cross section is considered tobe the center point of the cross section. The channel need not bestraight, and, consequently, different portions of the channel may havedifferent longitudinal axes. Further, in various embodiments,“substantially circular” cross sections are meant to denominate crosssections whose deviation from perfect circularity is characterized by aratio of the largest diameter to the smallest diameter of less than 1.3,preferably less than 1.1, even more preferably less than 1.03. Diametersare defined with respect to the center point of the cross section.

In one embodiment, the method includes providing a patterned wafer thatincludes at least one exposed electrically conductive region and atleast one exposed electrically insulating region; and electroplating aninverse channel portion with substantially semicircular cross sectiononto the wafer, thereby forming a first master mold. This first mastermold may then be employed to emboss, either directly or via two transfermolds, a channel portion in a polymer sheet. Subsequently, the polymersheet may be aligned and bonded with a second embossed polymer sheetthat contains a corresponding channel portion, i.e., a channel portionthat is substantially mirror symmetric to the channel portion in thefirst sheet, so that a channel with substantially circular cross sectionis defined between the two sheets. In some embodiments, the same mastermold may be used to emboss both sheets, and in other embodiments, asecond master mold, fabricated in a similar manner as the first mastermold, may be used. In one embodiment, channels are seeded with cells. Incertain embodiments, the channel is characterized by a graduallychanging diameter, and/or by a smooth and gradual bifurcation into twochannels.

The electrically conductive region may contain gold, chromium, titanium,tungsten, platinum, copper, silver, aluminum, or a combination thereof.The electrically insulating region may contain silicon dioxide and/orsilicon nitride. The patterned wafer may further include a substrate,e.g., containing or consisting essentially of silicon. In someembodiments, the electrically conductive region, which may be continuousor discontinuous, is deposited on the substrate, and the electricallyinsulating region(s) are deposited, at least in part, on theelectrically conductive region. In alternative embodiments, theelectrically insulating region is deposited on the substrate, and theelectrically conductive region(s) are, at least partially, depositedthereon. The electroplated inverse channel portion may comprise a metalselected from the group consisting of copper, gold, nickel, and/orsilver.

In certain embodiments, the method of producing the microfluidicstructure may be repeated to form additional microfluidic structures,and the resulting plurality of microfluidic structures may be stacked ina way that it defines a front surface containing the outlets of thechannels. The method may further be repeated to form a microfluidicheader, which may be coupled to the front surface and aligned with thechannel outlets so as to fluidically couple the microfluidic structurestogether.

In another aspect, embodiments of the invention feature a microfluidicstructure, which includes a polymer scaffold that defines a firstchannel therein. At least a portion of the channel is characterized by asubstantially circular cross section and a diameter less than 300 μm. Insome embodiments, the ratio of any first diameter of the channel to anysecond diameter that intersects with the first (i.e., lies in the samecross section) is less than 1.3, preferably less than 1.1. In oneembodiment, the channel is further characterized by a gradually changingdiameter. In some embodiments, the first channel bifurcates into secondand third channels, and the passages between the first and the secondchannels as well as between the first and the third channels are smoothand gradual. In certain embodiments, cells are seeded within thechannel(s).

In yet another aspect, embodiments of the invention feature amicrofluidic device. The device includes a plurality of microfluidicstructures and a microfluidic header coupling the structures together.Each microfluidic structure includes a polymer scaffold defining atleast one channel. The structures are stacked to form a stackedstructure that defines a front surface containing outlets of thechannels. The microfluidic header is coupled to the front surface andaligned with the outlets so as to fluidically couple the structurestogether. In some embodiments, the microfluidic header includes aplurality of polymer scaffolds, each defining a network of channels. Incertain embodiments, cells are seeded within the channels. At least aportion of one channel may be characterized by a substantially circularcross section and a diameter less than 300 μm.

In still another aspect, embodiments of the invention provide a methodfor fabricating a microfluidic structure that includes: providing apatterned wafer; isotropically etching the wafer with xenon difluorideto form a master mold with an inverse channel portion having asubstantially semicircular cross section; embossing the master mold intoa transfer mold; embossing the transfer mold into a polymer sheet; andaligning and bonding the polymer sheet with a substantially mirrorsymmetric second polymer sheet to define a channel with substantiallycircular cross section between the sheets. In certain embodiments, thediameter of the inverse channel changes gradually from a first to asecond diameter.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, aspects, features, and advantages ofthe invention will become more apparent and may be better understood byreferring to the following description taken in conjunction with theaccompanying drawings, in which:

FIG. 1 is a flow diagram illustrating methods of fabricatingmicrofluidic devices in accordance with various embodiments of theinvention;

FIG. 2A is a scanning electron microscopy (SEM) image of one embodimentof a copper electroplated mold on a silicon wafer showing four levels ofsmooth vessel bifurcations;

FIG. 2B is a higher magnification SEM image of the copper electroplatedmold on the silicon wafer of FIG. 2A showing an inverse half-cylindricalgeometry of the electroplated mold that may be used to embosshalf-cylindrical channels in a polymer sheet;

FIG. 3A is an SEM image illustrating a top view of one embodiment of apolystyrene film hard embossed using a copper electroplated mold;

FIG. 3B is an SEM image illustrating a side view of the polystyrene filmdepicted in FIG. 3A;

FIG. 4A illustrates a XeF₂-etched silicon tool for fabrication of apolystyrene microfluidic device in accordance with one embodiment of theinvention;

FIG. 4B illustrates a PDMS transfer mold for fabrication of apolystyrene microfluidic device in accordance with one embodiment of theinvention;

FIG. 4C illustrates an embossed polystyrene part for fabrication of apolystyrene microfluidic device in accordance with one embodiment of theinvention;

FIG. 5A is a photo of a sealed polystyrene microfluidic device withintegrated tubing in accordance with one embodiment of the invention;

FIG. 5B is an SEM photo of a channel cross section of the device of FIG.5A;

FIG. 6A is a flow diagram for fabricating a master mold according to apatterned gap electroplating process in accordance with one embodimentof the invention;

FIG. 6B is a schematic illustration of an exemplary copperelectroplating process in accordance with the method of FIG. 6A formicrochannel networks with varying diameters, in which the seed metal ispatterned so that subsequent electroplating contacts open new seed metalareas after a time delay, thereby controlling the feature height andwidth;

FIG. 6C is a perspective view of an exemplary structure fabricatedaccording to the method of FIG. 6A;

FIG. 7A is a flow diagram of a method for fabricating a master moldaccording to a buried electrode electroplating process in accordancewith one embodiment of the invention;

FIG. 7B is a schematic illustration of an exemplary copperelectroplating process in accordance with the method of FIG. 7A,starting with blanket seed metal deposition, patterning of alow-temperature oxide, electroplating of copper on open areas of theseed metal, and expanding isotropically as the plating time isincreased;

FIG. 7C is a perspective view of an exemplary structure fabricatedaccording to the method of FIG. 7A;

FIG. 8A is a flow diagram of a method for fabricating a master moldaccording to an electroplating process combining features of a patternedgap and a buried electrode process in accordance with one embodiment ofthe invention;

FIG. 8B is a perspective view of an exemplary structure fabricatedaccording to the method of FIG. 8A;

FIG. 8C is a perspective view of an exemplary electroplated channelfabricated from the structure shown in FIG. 8B;

FIG. 9A is an exploded view of a flow distribution manifold (header)ready for assembly with a vertical stack of flow networks in accordancewith one embodiment of the invention;

FIG. 9B illustrates the flow distribution manifold of FIG. 9A afterassembly;

FIG. 9C illustrates an assembly of microfluidic devices, incorporatingseven lateral flow layers with inlet and outlet headers, to form athree-dimensional integrated device in accordance with one embodiment ofthe invention;

FIG. 10 is a diagram illustrating cell adhesion, monolayer formation,and cell viability for primary human umbilical vein endothelial cells(HUVEC) seeded in a polystyrene network; and

FIG. 11 illustrates a calcein-AM live cell stain of primary HUVEC 18hours after seeding in open channels (200 μm diameter) using anelectroplating fabrication technique.

DESCRIPTION

Described herein are various embodiments of microfluidic structures andthree-dimensional microfluidic devices for biomedical applications.Computational models for microvascular networks, precise microfluidicfabrication technologies, and an endothelial cell model system may beintegrated to clearly establish functional cell phenotype in a truethree-dimensional construct. This technology may be used to manufacturedevices for in-vitro applications, including: fundamental vascularbiology research; investigations of clotting, inflammation, plaqueformation, and other pathologic states of vasculature; and drugdiscovery and drug safety testing. Moreover, the technology may betranslated into precision three-dimensional biodegradable scaffoldsintegrated with co-culture models and matrix materials suitable fortissue engineering applications, such as the development of wearable orimplantable devices that incorporate vascular access and amicrocirculation.

In accordance with various embodiments of the invention, microfluidicstructures containing substantially cylindrical microchannels ofvascular-like geometry, with diameters ranging from approximately 5microns to more than 10 mm and with lengths ranging from approximately100 microns to several centimeters, may be fabricated. Aspects of thevascular-like geometry may include smooth microchannel walls,substantially circular channel cross-sections, continuous changes inchannel diameters, and gradual transitions between channels atbifurcations. These features allow for smooth flow paths throughout theentirety of the channel network.

Microfluidic structures may be constructed using scaffolding materialscompatible with the seeding, expansion, and sustainment of functionalvascular cells, including endothelial cells and smooth muscle cells. Invarious embodiments of the invention, methods for fabricating suchmicrofluidic scaffolds involve fabricating master molds that mimic thegeometry of microvascular networks and embossing these master molds intoa well-established substrate for tissue culture. Certain methods mayproduce cylindrical geometries suitable for microvessels at diameters assmall as tens of microns, distinguishing them from other commonmicrofluidic fabrication techniques such as SU-8 photolithography orDeep Reactive Ion Etching.

The flow chart depicted in FIG. 1 provides an overview of methods forfabricating microfluidic structures according to various embodiments.These methods begin with the design and fabrication of one or morephotomasks defining structures corresponding to the desired channelnetwork (step 100). The mask layout may be created in a computerdrawing, and may then be converted into a Computer-Aided Design (CAD)layout, e.g., using a software package such as Tanner L-Edit. CADlayouts provide a format suitable for conversion into a photomaskgenerated by electron-beam lithography or a similar technique.

In step 102, the photomask is used to pattern a substrate, typically asilicon wafer. Patterning may generally involve several lithography,chemical development, etching, baking, and lift-off steps. Theparticular process sequence involved depends on the ensuing master moldfabrication steps, and is explained in detail further below in thecontext of specific methods.

In some embodiments, illustrated by the left-hand branch of the flowchart depicted in FIG. 1, the patterned wafer contains electricallyconductive regions and electrically insulating regions, and serves asthe seed structure for electroplating. During the electroplating step104, the patterned wafer is coupled to the cathode of an electriccircuit, and immersed in an electrolytic bath that also contains ametallic anode. The anode material, which may be, for instance, copper,nickel, gold, or silver, dissolves and deposits on the exposed,electrically charged metallic regions of the patterned wafer. Thereby, ahalf-relief for subsequent hard embossing is formed. A portion of anexemplary electroplated master mold with eight generations of bifurcatedmicrochannels having semi-circular cross-sections is shown in the SEMimages of FIGS. 2A and 2B. In particular, FIG. 2A shows five generationsof channels, and FIG. 2B shows a close-up of the bifurcation pointsbetween the channels. The bifurcation points show smooth transitionsthat allow non-disturbed flow through those regions.

The electroplated master mold may be used directly to hard embosssubstantially half-cylindrical features into a polymer sheet, such as apolycarbonate or polystyrene sheet (step 106). For cell cultures ofnumerous cell types, tissue culture grade polystyrene provides awell-established platform. In an exemplary hard embossing technique thatis robust, reproducible, and high-precision, polystyrene sheets fromPlaskolite, Inc. (040″ Clear UVF Styrene), cut to the appropriate size,are hard embossed using a metal-plated silicon mold as described above.More specifically, the silicon mold and polystyrene sheet are sandwichedbetween two pieces of Kapton film (Fralock Inc.) in an embossingapparatus, which allows for precise control of the temperature andpressure. The temperature of the apparatus may be raised to 130° C., andthe chamber evacuated, causing the piston of the embosser to exert apressure of 25 inches Hg on the mold and polystyrene substrate. Thetemperature may be maintained at 130° C. for 15 minutes, then theapparatus cooled to approximately 40° C. The mold and substrate may thenbe removed from the embosser and separated to form a free-standingpolystyrene device with rounded microchannels. Deeper channels thatallow for incorporation of flexible tubing may be created by attachingmetal tubing at the ends of the microchannels. An example of an embossedpolystyrene part is shown in FIGS. 3A and 3B.

In an alternative embodiment, the master mold may be used to emboss atransfer polymer mold (step 106′), resulting in a lowered relief, andthe transfer polymer mold used to emboss a second transfer polymer mold(step 106″), resulting in a raised relief. Finally, the second transferpolymer mold may be used to emboss the polystyrene sheet (step 106).This method is advantageous in that it avoids the repeated use of theelectroplated master molds, which can be fragile and delaminate overtime and temperature cycles. A transfer mold is often more robust sinceit is one solid piece, rather than a composite of a substrate with anelectroplated metal pattern on top. Suitable materials for both thefirst and second transfer mold are hard polymers that will withstandtemperature and maintain precise features over time and temperaturecycling, such as, e.g., polyurethane, polystyrene, or polypropylene.

Alternatively, and with reference to the right-hand branch of the flowchart depicted in FIG. 1, the patterned wafer may contain surfaceregions where the silicon is exposed, and regions protected by siliconoxide. Typically, a silicon wafer is coated with a silicon oxide layer,which is subsequently patterned lithographically. This wafer may beetched by xenon difluoride gas (XeF₂), which is highly selective toetching silicon, but not silicon oxide, and which removes silicon atomsin an isotropic fashion, i.e., etches vertically and laterally at thesame rate (step 108). Thereby, trenches with substantially semicircularcross sections are produced in the silicon wafer. Trenches with changingdiameters may be achieved by patterning the photoresist layer withfeatures of gradually changing widths, such as tapered features.

The pattern etched into the silicon wafer may be transferred to a masterpolymer mold, such as, for example, a PDMS mold or a polyurethane mold,thereby producing rounded raised structures similar to those of theelectroplated mold (step 110). The master polymer mold may then be usedto soft-emboss a polymer sheet, such as a polystyrene sheet, therebycreating rounded microchannel portions (step 112). In this case, thepolystyrene sheet may be heated to a higher temperature (150° C.) toallow it to form more readily around the soft polymer mold. In variousembodiments, producing a polymer copy of the etched silicon wafer ispreferable over using the etched silicon wafer directly since polymersmay exhibit material properties advantageous for biomedicalapplications, including transparency, low material cost,biocompatibility, and, in some cases, biodegradability. An exemplarysilicon wafer, polymer (e.g., PDMS) transfer mold, and embossedpolystyrene part created from this process are depicted in FIGS. 4A, 4B,and 4C, respectively.

In an optional step 114, the channel portions in the polystyrene surfacemay be treated chemically, e.g., with an oxygen plasma using a TechnicsMicrostripper with a setting of 100 mW and 100 mTorr O₂ pressure for oneminute, so that they become more hydrophilic and friendly to celladhesion. Two corresponding embossed, treated surfaces may then beplaced in contact, and the channels portions aligned with the aid of anoptical microscope such as to form substantially cylindrical channels(step 116). Flexible silastic tubing (Dow Corning Inc.) may be cut andplaced in the deeper channels designed for tubing connections. The twosheets may be thermally bonded in the embossing apparatus at 90° C. for30 minutes. The flexible tubing may also be sealed in place duringbonding, creating a seamless interface for injection of cells. Anexemplary microfluidic structure, fabricated by hard embossing anelectroplated master mold into two polystyrene sheets, is depicted inFIG. 5A. The close-up of FIG. 5B shows the circular cross section of achannel outlet. Since the structure depicted in FIGS. 5A and 5B exhibitssubstantial mirror symmetry, the same master mold may be used to embossboth polymer sheets. In general, however, the fabrication of twocorresponding sheets whose channel portions can be lined up requires twomaster molds with substantially mirrored reliefs.

In step 118, the microfluidic structure may be plated with cells ofvarious types, e.g., with endothelial cells. Tissue culture gradepolystyrene, which serves as the industry standard for cell and tissuearrays for high-throughput screening and other laboratory applications,is highly suitable for establishing a robust functional phenotype.

A. Electroplating Methods for Fabricating Master Molds

Various electroplating methods may be employed to fabricate master moldswith inverse channel portions that feature substantially semicircularcross sections, gradually varying diameters, and/or smooth bifurcations.FIGS. 6A-6C illustrate a metal electroplating process known as a“patterned gap” process, which is capable of producing silicon mastermolds with a range of microchannel diameters and smooth flow transitionsat bifurcations and between successive generations of channels. Asdetailed in the flow diagram of FIG. 6A and the schematic of FIG. 6B,the process begins with the deposition of a blanket layer ofelectrically insulating material onto a clean silicon wafer or othersubstrate (step 600). For example, a thin silicon oxide or siliconnitride layer may be produced by thermal oxidation. This layer may bebetween approximately 0.5 and 1 μm thick.

In the subsequent steps, the insulating layer may be patterned accordingto the desired microfluidic structure, utilizing the photomaskfabricated in step 100. In one embodiment, the insulator-coated wafer iscovered with a specialized ‘lift-off’ resist (e.g., Microchem, Corp. LORseries) and the photoresist AZ1518 (step 602). The photomask is thenplaced onto the wafer, e.g., using a contact aligner (e.g., Karl SussMA-6), for the subsequent lithography step 604. After illumination, thephotoresist and lift-off resist may be developed using diluted AZ400Kand MF319, respectively (step 606). In step 608, the seed metal forelectroplating is deposited. Suitable seed metals include chrome, gold,titanium, tungsten, and platinum, as well as combinations thereof, suchas chrome-gold (e.g., 1000 A Cr/3000 A Au) or titanium-tungsten. In someembodiments, the seed metal layer includes an adhesion layer of chrome,titanium, or tungsten-titanium, and a top metal layer of gold, platinum,or copper. The seed metal may be deposited in a sputterer (e.g., MillLane DC Magnetron Sputterer) or in an evaporator (e.g., Denton E-BeamEvaporator). Lift-off (step 610) may be achieved by ultrasonicallyagitating the wafer in a solvent. The resulting structure 640, depictedin FIGS. 6B and 6C, includes a substrate 650, a continuous insulatinglater 652, and metallic regions 654 on top of the insulating layer 652.In regions 656 where the metal has been lifted off, the insulating layer652 is exposed. The patterned wafer 640 is ready for metalelectroplating.

With reference to FIGS. 1 and 6A, copper electroplating, at step 104,may then be achieved by submerging the wafer in a commercially-availablebath solution (Technic, Inc. Copper Bath RTU) and applying properelectrical current within the bath. By optimizing the application of thecurrent (e.g., 70 mA measured with a Fluke volt meter), a reliablecopper growth rate of 15 μm/hr may be achieved. Alternatively, othermetals such as, for example, nickel, gold, and silver may beelectroplated.

In one embodiment, the metal electroplating begins immediately inregions for which the metal pattern is directly connected to the anode,but is delayed in other regions by the presence of the gaps breaking theconnection between the anode and the exposed seed metal. The length ofthese gaps governs the delay time prior to the start of electroplatingthe metal onto the next level of microchannels with the network design.For instance, a gap of 100 microns introduces a time delay such that thelargest ridges are 100 microns taller than the next level structures. Byvarying the gaps in a systematic fashion, an entire range of microvesseldiameters may be achieved on embossed layers. For example, FIG. 6Cillustrates three exemplary metal patterns 670, 672, and 674. Patterns670 and 674 each comprise three straight metal portions, whereas pattern672 comprises a straight metal portion, and two branching metalportions. If the leftmost metal portions are connected to the anode,metal deposits on these portions first. Once a certain thickness of thedeposited layer is reached, the deposited metal forms an electricalconnection with the next portions to the right, and metal depositsthereafter in both regions. As a result, the diameter of the depositedstructure changes at the location of the gap. Patterns 670 and 674 bothresult in an inverse channel whose diameter changes twice; pattern 672results in an inverse channel that branches into two channels of smallerdiameter. Patterned gap electroplating produces rounded structures,which, when embossed into a polymer sheet, achieves rounded channelportions. The exact shape of the channel cross section depends on theplated material, the dimensions of the seed metal structures, andelectroplating parameters. In some embodiments, cross sections resembletrapezoids, in others, they approximate semicircles.

An alternative method, which may reproducibly achieve nearly circulargeometries, is an electroplating process known as a “buried electrode”process, depicted in FIGS. 7A-7C. In one embodiment, this processinvolves, in step 700, depositing a blanket metal layer, such as achrome-gold layer (e.g., 500 A Cr/1000 A Au), on a silicon wafer byevaporation or sputtering. In step 702, an insulating layer (e.g., anoxide layer) may be deposited on top of the seed-metal, using, forinstance, plasma-enhanced chemical vapor deposition (PECVD; OxfordSystem 100 PECVD Reactor). The thickness of the oxide layer may be inthe range of approximately 0.5 to 1 μm. In one embodiment, the oxide ispatterned, in steps 704 and 706, using a contact lithography system(Karl Suss MA-6) and Shipley 1822 photoresist (MF319 developer). Thisstep may be followed, in step 708, by wet-etching of the oxide inhydrofluoric acid (Buffered Oxide Etch DI 1:7 HF) or by reactive ionetching. After the oxide is etched, the photoresist may be removed witha solvent (SVC-14) (step 710), making the wafer ready for metalelectroplating (step 104).

An exemplary patterned structure 740, depicted in FIGS. 7B and 7C,includes, in this order, a substrate 750, a continuous electricallyconducting layer 752, and electrically insulating regions 754. Inregions 756 where the insulating layer has been etched, the conductinglayer 752 is exposed. However, in contrast to the patterned gapstructure 640 depicted in FIG. 6C, the metallic regions 752 in structure740 are below the insulating regions 754. This feature eliminatesdelamination issues that may occur in the patterned gap process, and,consequently, alleviates the optimization of process parameters for thedesired channel geometries.

In general, the shape and smoothness of the electroplated reliefstructures depend on the dimensions of the wafer pattern, as well as onvarious electroplating process parameters, including the electroplatedmaterial, the current density, the anode configuration, and the bathconcentration and agitation. Nearly perfectly inverse circulargeometries in master molds may be achieved in a commercial copper bathsolution (e.g., Technic, Inc. Copper Bath RTU) without agitation at acurrent density in the range from 5.5 mA/cm² to 7.75 mA/cm². To obtainuniform current densities, an anode in spiral wire configuration may beemployed. The resulting plating rate is about 14 μm/hour. Starting withfeature widths of 50 μm in the patterned wafer, the height of theinverse channel portions approaches the width at a diameter of theinverse channel portion of about 200 μm. Using the ratio of the largestto the smallest diameter of realistic channels as a measure of thedegree of circularity, ratios of less than 1.3 can be achieved. FIG. 3B,for example, shows a semicircular channel with a larger diameter of199.1 μm and a smaller diameter of 176.8 μm, corresponding to a ratio of1.12. Some embodiments may result in ratios of less than 1.1, or even ofless than 1.03.

The buried electrode wafer depicted in FIG. 7C, while enablingsubstantially semicircular inverse channel portions, does notsimultaneously provide for changes in the diameter. Since the seed metallayer 752 is continuous, the electroplated material deposits in allexposed regions at the approximately the same rate. Therefore, if theetched patterns were to include tapered features in order to vary thewidth of the inverse channel portions, the height of the inverse channelportions would not vary. Consequently, the cross sections of the inversechannel portions would not be semicircular throughout.

In order to employ the buried electrode process and to achieve inversechannel portions with varying diameter, the advantageous properties ofthe buried electrode and the patterned gap process can be combined in ahybrid method illustrated in FIGS. 8A-8B. This method employs thepatterning of both the seed metal and the insulating layers. In detail,as shown in FIG. 8A, the method involves, in a first step 800, coating asubstrate (e.g., silicon) with lift-off resist and photoresist, andlithographically patterning it (similarly to steps 602 and 604 in FIG.6A). Then, in step 802, the seed metal layer is deposited on the waferby evaporation or sputtering (similarly to step 700 in FIG. 7A). In thesubsequent lift-off step 804 (resembling step 610 of FIG. 6A), the metalis removed in an agitated solvent bath in certain areas where it isdesired to change the channel diameter. The patterned wafer issubsequently coated with an insulating layer (step 702), such as siliconoxide, and with a photoresist layer (step 704), and patterned by meansof lithography (step 706) and etching (step 708). After removal of theresist and cleaning of the wafer (step 710), the wafer is ready forelectroplating (step 104).

An exemplary wafer 840, patterned according to the method shown in FIG.8A, is depicted in FIG. 8B. It contains, in this order, a substrate 850,a seed metal layer 852, and an insulating layer 854. Along the axis ofthe channel structures (x-direction), the metal layer is patternedsimilarly to structure 640; along the perpendicular y-axis, the metallayer is continuous. Gaps 856 in the metal layer may be filled withinsulating material. The discontinuity of the seed metal layer resultsin diameter changes of the raised structures produced by electroplating.The insulating layer 854 is patterned, in the y-direction, like that ofthe buried electrode wafer 740, and enables the fabrication ofsubstantially semicircular channel portions. FIG. 8C illustrates theshape of an electroplated structure that results from the portion 870 ofthe patterned wafer 840.

B. Integrated Three-Dimensional Microfluidic Networks

Microfluidic scaffolds having two layers of polymer sheets joinedtogether typically form a two-dimensional flow network. In order tobuild a three-dimensional flow network, several such two-dimensionalstructures may be combined. For example, as illustrated in FIGS. 9A-9C,several individual microfluidic structures 900 may be stacked to form astacked structure 902. The individual layers 900 of the stackedstructure 902 may then be fluidically connected, e.g., with tubingfitted into the channel outlets 904. Alternatively, the structures 900may be integrated into a three-dimensional flow network using amicrofluidic header 906 with smooth, curved flow paths that distributesfluid evenly through the stacked structure 902.

The header 906 may be fabricated using stereolithography (SLA)technology, or employing the techniques described earlier. For example,FIG. 9A illustrates a header 906 containing two parts with correspondinghalf-channel flow networks, which may be produced by embossingelectroplated reliefs into blocks of polystyrene as earlier described.To integrate the individual layers 900, they may be coupled (e.g.,snapped) together and placed in direct contact with the inlet and outletheaders 906, as depicted in FIG. 9B. Top and bottom plates 908 may beused to arrest the relative positions of headers 906 and stackedstructure 902.

In one embodiment, the microchannel flow network of the headers 906 isrotated with respect to the flow networks in the stacked structure 902,and aligned with the outlets 904 in a front surface 910 of the stackedstructure 902 that arises from a flush arrangement of the layers 900. Ifthe outlets 904 are placed along a straight line, as depicted, theheader 906 need only contain a single channel flow network. Generally,structures 902 may contain different channel flow networks, whoseoutlets form a two-dimensional pattern in the front surface 908. Fluidicconnections between the layers 900 may then be achieved with a header906 that comprises several layers with microchannel networks itself.

A photograph of an exemplary assembly of a three-dimensional flownetwork is shown in FIG. 9C, with a single closed two-dimensional flownetwork (i.e., two half-channel layers fitted together) placed to theright-hand side for illustrative purposes. In this assembly, the headerchannels bifurcate with smooth flow paths in a vertical manner toconnect to each of the seven two-dimensional flow layers.

C. Biomedical Applications

Establishment of a robust functional phenotype for endothelial cells inthree-dimensional microfluidic constructs typically requires exquisitecontrol over numerous parameters associated with the cellmicroenvironment. Among these are surface chemical interactions betweenthe cultured endothelial layer and the walls of the scaffold, and thestability of the surface over time. In addition, the mechanical forcespresent must be well-controlled and stable over time and as a functionof cell seeding and local flow conditions.

As described herein, microfluidic devices according to variousembodiments provide microchannels with smooth walls, substantiallycircular cross sections, and gradually varying diameters. Thecombination of these features results in smooth transitions atbifurcations. By contrast, bifurcations of channels with rectangularcross sections typically result either in a change of the channel aspectratio, i.e., the ratio of channel width to channel height, or, if thearea of the channel cross section changes at the bifurcation while theaspect ratio remains constant, in abrupt changes in channel height. Bothchanges in the aspect ratio and the sharp comers associated with abruptchanges in channel dimension typically result in flow turbulences.Devices manufactured in accordance with certain embodiments of thepresent invention, on the other hand, feature smooth flow paths and, asa result, well-controlled forces, throughout the entirety of the channelnetwork.

Chemical interactions between endothelial cells and the channelsinfluence the ability of the cells to attach as a confluent monolayer tothe network channel walls and maintain viability under normal cultureconditions. As depicted in FIG. 10, cell attachment and monolayerformation within polystyrene networks micromolded fromstereolithographic masters has been achieved, and is best visualizedusing phase contrast imaging. Cell viability has been confirmed using acalcein AM live cell stain in which only viable cells fluoresce. In someembodiments, both cell attachment/monolayer formation and viability areconsistent among channels and independent of the channel diameter.Overall, these results show that primary HUVEC attach and form aconfluent monolayer within three-dimensional networks, and remain viableafter 24 hours of culture, validating the fabrication approach describedabove for generating three-dimensional microfluidic devices formicrovascular network applications.

Open polystyrene half-channels having diameters of about 200 μm,generated with the above-described electroplating fabrication approach,may be plated for 18 hours with HUVEC and assessed for monolayerformation and cell viability. As shown in FIG. 11, primary HUVEC formedan intact monolayer and stained with calcein AM (a marker of cellviability), thus validating the compatibility of the electroplatingfabrication process proposed above with endothelial cell culture.

Several advantages to the approaches described herein include theability to produce vascular networks having vessels with substantiallycylindrical geometries, and to construct smooth transitions at vesselbifurcations and vessel diameter changes in a manner similar to healthyphysiologic structures. Once such a healthy physiologic structure hasbeen manufactured as described herein, a specific geometry associatedwith disease or pathology may be introduced into a single one of thevessels and studied in isolation. For example, a rectangular geometry, asharp angle or bend, a dead space, and/or a sudden expansion orcontraction at a vessel bifurcation or at a dimensional change in thevessel may be introduced therein to approximate an atherosclerotic orother cardiovascular condition. In such a fashion, a disturbed flowcondition associated with, for example, thrombus formation, plaqueformation, or an inflammatory response may be studied in isolation andin a controlled fashion at that single diseased or pathologic structure.

Having described certain embodiments of the invention, it will beapparent to those of ordinary skill in the art that other embodimentsincorporating the concepts disclosed herein may be used withoutdeparting from the spirit and scope of the invention. For example, theelectroplating methods described may also be applied to the optimizationof inverse channel geometries with non-semicircular cross sections.Accordingly, the described embodiments are to be considered in allrespects as only illustrative and not restrictive.

1. A method for fabricating a microfluidic structure, comprising: (a)providing a patterned wafer comprising at least one exposed electricallyconductive region and at least one exposed electrically insulatingregion; (b) electroplating an inverse channel portion with substantiallysemicircular cross section onto the wafer, thereby forming a firstmaster mold; (c) employing the first master mold so as to emboss achannel portion in a first polymer sheet; (d) aligning and bonding thefirst polymer sheet with a second polymer sheet having a correspondingchannel portion such as to define a first channel with substantiallycircular cross section between the polymer sheets.
 2. The method ofclaim 1 further comprising employing the first master mold so as toemboss a channel portion in the second polymer sheet.
 3. The method ofclaim 1 further comprising repeating steps (a) and (b) to form a secondmaster mold having an inverse channel portion substantially mirrorsymmetric to the channel portion on the first master mold, and employingthe second master mold so as to emboss a channel portion in the secondpolymer sheet.
 4. The method of claim 1 wherein step (c) comprisesdirectly embossing the master mold into the first polymer sheet.
 5. Themethod of claim 1 wherein step (c) comprises embossing the master moldinto a first transfer mold, embossing the first transfer mold into asecond transfer mold, and embossing the second transfer mold into thefirst polymer sheet.
 6. The method of claim 1 wherein, for any firstdiameter of the channel and any second diameter of the channelintersecting with the first diameter, the ratio of the first diameter tothe second diameter is less than 1.3.
 7. The method of claim 1 whereinthe channel is characterized by a gradually changing diameter.
 8. Themethod of claim 1 wherein the first channel bifurcates into at least asecond channel and a third channel, a first passage between the firstchannel and the second channel and a second passage between the firstchannel and the third channel being smooth and gradual.
 9. The method ofclaim 1 wherein the electrically conductive region comprises a metalselected from the group consisting of chromium, gold, titanium,tungsten, platinum, silver, aluminum, and copper.
 10. The method ofclaim 1 wherein the electrically insulating region comprises a materialselected from the group consisting of silicon dioxide and siliconnitride.
 11. The method of claim 1 wherein the patterned wafer furthercomprises a substrate, and wherein the electrically conductive region isdeposited on the substrate, and the electrically insulating region isdeposited on the electrically conductive region.
 12. The method of claim11 wherein the substrate comprises silicon.
 13. The method of claim 11wherein the electrically conductive region is continuous.
 14. The methodof claim 11 wherein the electrically conductive region is discontinuous.15. The method of claim 1 wherein the patterned wafer further comprisesa substrate, and wherein the electrically insulating region is depositedon the substrate, and the electrically conductive region is deposited onthe electrically insulating region.
 16. The method of claim 1 whereinthe electroplated inverse channel portion comprises a metal selectedfrom the group consisting of copper, nickel, silver, and gold.
 17. Themethod of claim 1 further comprising: (e) optionally repeating steps (a)and (b), and repeating steps (c) and (d) to form a plurality ofmicrofluidic structures; (f) stacking the plurality of microfluidicstructures to form a stacked microfluidic structure that defines a frontsurface comprising outlets of the channels; (g) repeating steps (a),(b), (c), and (d) to form a microfluidic header; and (h) coupling themicrofluidic header to the front surface defined by the stackedmicrofluidic structure, and aligning the microfluidic header to theoutlets of the channels so as to fluidically couple the microfluidicstructures together.
 18. The method of claim 1 further comprisingseeding cells within the channel.
 19. A microfluidic structure,comprising: a polymer scaffold defining a first channel therein, atleast a portion of the first channel being characterized by asubstantially circular cross section and a diameter less than 300micrometers.
 20. The microfluidic structure of claim 19 wherein, for anyfirst diameter of the channel and any second diameter of the channelintersecting with the first diameter, the ratio of the first diameterand the second diameter is less than 1.3.
 21. The microfluidic structureof claim 19 wherein, for any first diameter of the channel and anysecond diameter of the channel intersecting with the first diameter, theratio of the first diameter and the second diameter is less than 1.1.22. The microfluidic structure of claim 19 wherein the first channel isfurther characterized by a gradually changing diameter.
 23. Themicrofluidic structure of claim 19 wherein the first channel bifurcatesinto at least a second channel and a third channel, a first passagebetween the first channel and the second channel and a second passagebetween the first channel and the third channel being smooth andgradual.
 24. The microfluidic structure of claim 19 further comprisingcells seeded within the first channel.
 25. A microfluidic device,comprising: (a) a plurality of microfluidic structures, each structurecomprising a polymer scaffold defining at least one channel therein, theplurality of microfluidic structures being stacked so as to form astacked microfluidic structure that defines a front surface comprisingoutlets of the channels; and (b) a microfluidic header comprising atleast one polymer scaffold that defines a network of channels therein,the microfluidic header being coupled to the front surface of thestacked microfluidic structure and aligned with the outlets of thechannels so as to fluidically couple the microfluidic structurestogether.
 26. The microfluidic device of claim 25 wherein themicrofluidic header comprises a plurality of polymer scaffolds, eachpolymer scaffold defining a network of channels therein.
 27. Themicrofluidic device of claim 25 further comprising cells seeded withinthe channels.
 28. The microfluidic device of claim 25 wherein at least aportion of at least one channel is characterized by a substantiallycircular cross section and a diameter less than 300 micrometers.
 29. Amethod for fabricating a microfluidic structure, comprising: (a)providing a patterned wafer; (b) isotropically etching the wafer withxenon difluoride so as to form a master mold defining an inverse channelportion with substantially semicircular cross section; (c) embossing themaster mold into a transfer mold; (d) embossing the transfer mold into afirst polymer sheet; and (e) aligning and bonding the first polymersheet and a second polymer sheet having a corresponding channel portionsuch as to define a first channel with substantially circular crosssection between the polymer sheets.
 30. The method of claim 29 wherein adiameter of the inverse channel portion changes gradually from a firstdiameter to a second diameter.